Abstract 932: Translated Alternative Isoforms Coincide With Disordered Sequences in the Cardiac Proteome

Abstract

Introduction: Alternative splicing is widely implicated in cardiac development and diseases, but few isoform transcripts have ever been empirically observed as translated proteins. We aim to understand how alternative splicing diversifies proteome function. Method: We show an RNA-seq guided proteomics approach, comprising a custom pipeline of sequence read alignment, splice junction determination, and in silico filtering and translation to identify alternative isoforms across multiple cell types and anatomical regions of the human heart. We further applied the method to a time course study of human iPSC differentiation into cardiomyocytes, collecting isotope-tagged quantification data on a Q-Exactive HF mass spectrometer to examine isoform expression patterns during myocyte differentiation. We cataloged the translated non-canonical isoforms in the heart, and characterized sequence features upon which the empirically observed alternative sequences impinge. Result: We identified 1,216 distinct non-canonical isoform sequences at 1% FDR, including 143 sequences from 107 genes not found in the comprehensive databases TrEMBL and RefSeq. We observed tissue-specific and differentiation stage-specific differences in isoform expression patterns. The data show a proteome-wide enrichment of translated alternative isoforms sequences at intrinsically disordered regions of proteins with implication on isoform function. We observed examples where protein PTMs may be modulated by the exclusion of modification sites via splicing including a novel isoform we found in MYBPC3 which coincides with PKA-mediated phosphorylation implicated in diastolic function regulation in animal models. Conclusion: This is the first study to combine experimental RNA-seq and proteomics to investigate how splicing impacts the human cardiac proteome. The approach may avail understanding of the functional outcome of splicing and its role in disease mechanisms.